It has long been known that cell shape regulates growth control and anchorage dependence in normal (untransformed) cells, and that transformed cells exhibit altered morphology and matrix attachment properties. The shape of a cell is determined primarily by its internal actin cytoskeletal architecture and influenced by focal adhesions, which constitute points of linkage between the extracellular matrix (ECM), the integrin receptor system land internal signaling networks. HEF1, and the related proteins pl30Cas and Efs/Sin, define the Cas (Crk-associated substrate) family of signaling proteins. All members of this family reside at focal adhesions in interphase cells, and function in part as scaffolds to assemble complexes of proteins that transmit signals originating through integrins. Initial studies of these proteins focused in large part on defining their roles in the processes of cell adhesion and cell migration, leading to the determination that members of this family are important transducers of integrin signaling in these processes. However, our work in the last period of this grant has identified a number of HEF1-specific functions that suggest unique activities for this protein as a sentinel of cell adhesion status in apoptosis. Further, recent work has strongly suggested that the cleavage and relocalization of HEF1 at mitosis is important for appropriate progression through cytokinesis, and that this function of HEF1 involves regulation of the activation cycle of the RhoA GTPase. Based on these and other results discussed herein, we propose that HEF1 has a unique function as a regulator of cell cycle progression through mitosis and cytokinesis, and that this effect is separable from its roles in attachment-related survival signaling. To investigate this idea, we propose three aims. We will test the hypothesis that HEF1 directly interacts with the RhoA-GEF protein ECT2/Pebble to activate RhoA in mitosis. We will evaluate the action of HEF1 at centrosomes, testing a complementary hypothesis that HEF1 controls mitotic progression by regulating centrosomally-associated regulators of M-phase progression. Finally, we will determine the long-term consequences of HEF1 deregulation for genomic instability in cancer.